Delayed immune reconstitution, and in particular T cell deficiency, is an important contributor to morbidity and mortality in allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients, leading to infections and malignant relapse. Strategies to enhance post-transplant T cell reconstitution can significantly improve the overall outcome of allo-HSCT. However, at the present time such strategies are not readily available. In the first funding period we have performed preclinical studies regarding administration of interleukin-7 (IL-7) or keratinocyte growth factor (KGF), as well as inhibition of sex steroids (chemical or surgical) resulting in three clinical trials at our center. In the current (second) funding period we have performed studies in mouse HSCT models demonstrating the feasibility and efficacy of adoptive cell therapy with ex vivo generated precursor T cells (preT) to enhance post-transplant T cell reconstitution. We found that adoptively transferred preT can be used as an off-the-shelf cell therapy and administered across MHC barriers to enhance thymic regeneration and T cell immunity. We are currently preparing a phase I clinical trial to study the adoptive transfer of third party cord blood-derived preT transduced with an anti-CD19 chimeric antigen receptor for allo-HSCT recipients with high risk ALL. Our proposal for the next funding period aims to further explore the potential benefits of adoptive transfer of preT, as well as modify our preT cell culture method to develop an artificial implantable hematopoietic niche that can support T cell development. We hypothesize that (a) preT rapidly reconstitutes T cell immunity in allo-HSCT recipients via both thymic and extrathymic developmental pathways, (b) preT can be engineered to enhance anti-tumor activity and the regeneration of thymic and extrathymic niches and (c) the preT culture system can be modified to develop an artificial hematopoietic niche for T cell development. We propose to study in Aim 1 the contribution of extrathymic sites to preT-derived T cell reconstitution, the crosstalk of preT and thymic stroma resulting in long-term thymic regeneration, and strategies to enhance preT transfer. In Aim 2 we propose to study genetic engineering of preT to enhance their anti-tumor or (extra)thymic regenerative potential. We also plan to engineer an artificial hematopoietic niche for T cell development that can be implanted to enhance T cell development.